1. Field of the Invention
This invention relates to a diagnostic assay for distinguishing closely related bacterial strains or species from one another. The assay takes advantage of the presence of highly conserved insertional elements which are uniquely located in the genomic or plasmid DNA of a given strain or species.
2. Description of the Prior Art
The bacterial genus Brucella has been identified as the causative agent of a number of diseases in humans and animals. It is responsive for economically important diseases in cattle and other livestock. Perhaps the most noteworthy of such diseases is bovine brucellosis, which is asssociated with abortions and infertility in cattle. This disease is caused by the gram-negative organism, B. abortus. Symptoms of equine brucellosis, also caused by B. abortus, include abortions and swelling or abscesses that form at the withers and poll. In swine, B. suis causes abortions in sows and is responsible for orchitis in boars. In goats, B. melitensis is frequently shed in the milk and thereby transmitted to humans. In sheep, B. ovis is responsible for ram epididymitis in males and occasional abortions in females. Other species of Brucella include B. neotomae (isolated from the desert wood rat) and B. canis, found in dogs. The taxonomy of Brucella also accounts for eight recognized biotypes (biovars) of B. abortus plus the vaccine strain B. abortus S19. There are also five biotypes of B. suis and three biotypes of B. melitensis.
Identification of an organism as belonging to the genus Brucella can be accomplished by observation of growth characteristics in combination with serological and bacteriological methods. Typing of the organism to establish species and biovar is determined by host range, susceptibility to lysis by phages, and by oxidative metabolic profiles. It was reported by Allardet-Servent et al. [J. Bacteriol. vol. 170:4603-4607 (1988)] that electrophoretic profiles of total genomic DNA reveal species-specific DNA fingerprints for B. abortus, B. melitensis, and B. suis, and that DNA fingerprints of B. ovis separated this species from all others. Ficht et al. [Advances in Brucellosis Research, pages 36-51 (1990)] report that RFLPs in Brucella chromosomal DNA at the omp 2 porin gene locus can be used to distinguish two classes of B. abortus.
In recent years bacterial genomes and plasmids have been reported to contain insertion sequences (ISs). The bacterial sources of many such ISs are given in Galas et al. [Mobile DNA, ed. Douglas E. Berg and Martha M. Howe, American Society for Microbiology, Washington, D.C., pages 109-115 (1989)]. ISs are highly specialized genetic elements which are transposable and have a highly variable genetic map location, even within populations of the same bacterial species [Brahma et al., J. Gen. Micro., vol. 128:2229-22234 (1982); Hartl et al., Philos. Trans. R. Soc. London B. Bio. Sci., vol. 312:191-204 (1986)]. They are typically from 0.75 to 2.5 kilobase pairs in length and are usually flanked by short perfect or nearly perfect inverted repeat sequences between 15 and 25 base pairs (bp) long. They can have a copy number in bacterial genomes of up to a few hundred per genome. ISs cause insertion mutations and other chromosomal rearrangements. Most ISs have one long open reading frame encoding a transposase. IS1, the smallest active transposable element known to exist in prokaryotes, relies upon an alternative mechanism for transposition; namely, the expression of a fusion protein that has IS1 transposase activity by frameshifting [Proc. Natl. Acad. Sci. USA, vol. 86:4609-4613 (1989)].
Thierry et al. [Nucleic Acids Research, vol. 18:188 (1990)] report that insertional sequences have been found in most mycobacteria which have been examined, and these elements are useful as taxonomic markers. Vary et al. [J. Clin. Micro., vol. 28: 933-937 (1990)] report that DNA probes which hybridize to a mycobacterial insertion sequence, IS900, were highly specific for Mycobacterium paratuberculosis. Vary et al., supra, also report on the use of DNA sequences derived from IS900 to prepare DNA primers for detection and identification of M. paratuberculosis by the polymerase chain reaction (PCR).